L-tryptophan, an essential amino acid, has been widely used as a feed additive, a raw material for medical drugs such as infusion solutions, and a material for healthfoods, and has been produced by chemical synthesis, enzymatic reaction, fermentation, etc.
Recently, production of L-tryptophan is mainly carried out by microbial fermentation. In the initial stage of industrialization, analogue resistant strains obtained by chemical mutation have been mainly used. However, as gene recombination technologies rapidly developed in the 1990s and regulatory mechanisms were understood at the molecular level, the recombinant E. coli and Corynebacterium strains obtained by genetic engineering techniques have been mainly used.
The production of tryptophan by microorganisms starts with DAHP(3-deoxy-D-arobino-heptulosonate-7-phosphate) produced by the polymerization of PEP (PhospoEnolPyruvate) that is an intermediate of glycolysis, with E4P (erythrose-4-phosphate) that is an intermediate of the pentose phosphate pathway. Then, tryptophan is biosynthesized from chorismate through the common aromatic biosynthetic pathway. Specifically, tryptophan is synthesized by anthranilate synthase (EC 4.1.3.27) encoded by trpE gene, anthranilate synthase (EC 4.1.3.27) and anthranilate PRPP transferase (EC 2.4.1.28) encoded by trpD gene, indole-3-glycerol phosphate synthase (EC 4.1.1.48) and phosphoribosylanthranilate isomerase (EC 5.3.1.24) encoded by trpC gene, and tryptophan synthase (EC 4.2.1.20) encoded by trpB gene and trpA gene. The gene cluster trpEDCBA that mediates the above reaction is placed in the chromosome and have an operon structure containing a single regulatory region.
A tryptophan operon is actively transcribed so as to produce a sufficient amount of tryptophan required by the cell. However, if tryptophan level in the cell is high, a repressor binds to tryptophan and then the tryptophan operon is inactivated by the binding of the repressor to operon regulatory region, thereby the transcription is inhibited.
In addition, operons for biosynthesis of amino acids such as threonine, phenylalanine, leucine, tryptophan and histidine have another regulatory mechanism known as an attenuation (J Bacteriol. (1991) 173, 2328-2340). As is known in the art with respect to the attenuation, under conditions deficient in amino acids, the structure of the mRNA corresponding specific sequence region between the promoter and the first gene of the operon on the chromosome, changes to a structure advantageous for the translation process to promote the expression of biosynthetic genes, but under conditions rich in the amino acids, the short transcribed mRNA forms a three-dimensional structure, named “hairpin structure, to inhibit the translation process (J Biol Chem., (1988) 263:609-612).
In the initial stage of the development of L-tryptophan-producing strains, it was a major object to increase the efficiency of production through the enhancement of enzyme activity either by releasing the feedback inhibition of tryptophan biosynthesis pathway enzymes caused by the final product, tryptophan or by increasing the copy number of the tryptophan operon genes on the chromosome or in the form of vector in order to enhance the expression of tryptophan biosynthetic enzymes (Appl. Environ. Microbiol., (1982) 43:289-297; Appl. Microbiol. Biotechnol., (1993) 40:301-305; Trends Biotechnol., (1996) 14:250-256).
Methods for imparting the ability to produce L-tryptophan to microorganisms include a method of imparting resistance to tryptophan analogues or anthranilate as the intermediate product by chemical mutation, or a method of modifying microorganisms by genetic engineering. Examples of the chemical mutation method include those described in Korean Patent Registration No. 1987-0001813, Korean Patent Registration No. 0949312 and the like, and examples of the modification method based on genetic engineering include various approaches which use a strain obtained by enhancing the transketolase-encoding tktA gene or the galactose permease-encoding galP gene in the aromatic amino acid biosynthesis pathway to increase the supply of E4P (erythrose4-phosphate) or PEP (phosphoenolpyruvate) and reducing the feedback inhibition of DAHP (3-deoxy-D-arabino-heptulosonate-7-phosphate) in order to enhance the aromatic biosynthetic pathway (Trends Biotechnol., (1996)14:250-256, Microbial Cell Factories (2009) 8:19), or a strain obtained by additionally introducing tryptophan operon genes into the vector or chromosome (Appl. Environ. Microbiol., (1982) 43:289-297, Appl. Microbiol. Biotechnol., (1993) 40:301-305).
However, even though the tryptophan operon was introduced with releasing the feedback inhibition of the biosynthetic enzymes, those approaches did not reached to an increase in the production yield of tryptophan, due to the regulatory mechanisms such as the inhibition or attenuation of the operon genes at transcription level.